ABSTRACT Despite widespread use, the only licensed tuberculosis (TB) vaccine available for human use, M. bovis Bacille Calmette-Guerin (BCG), has not been able to prevent TB from becoming a global health emergency. To achieve the 2030 targets of the WHO End TB Program, a new TB vaccine strategy is needed. For optimal effect, such a strategy will need to offer both improved efficacy following vaccination of TB-nave infants, as well as vaccine efficacy in the population of M. tuberculosis (Mtb)-exposed adolescents/adults in endemic regions. Advances in our understanding of TB immunology and vaccinology allow for a more tailored vaccine designed for this dual function. Based on specific antigen selection and adjuvanted administration, we have developed a novel single vaccine with dual use as a co-vaccine alongside BCG for nave individuals and as a standalone vaccine for previously exposed/infected individuals. This designed hybrid protein subunit vaccine (ESX1-Vax) is composed specifically of secreted ESX-1 related antigens that are missing from BCG, including ESAT-6 whose targeting is uniquely protective against re-growth of pre-existing infection. The hybrid protein is delivered with the CAF01 adjuvant that is highly effective in promoting T cell responses with defined TB-protective phenotype and function. Co-administration of ESX1-Vax/CAF01 with BCG does not hinder BCG effects. Instead, it acts synergistically to complement BCG vaccination by priming differentiation-resistant T cells targeting ESX-1 virulence factor antigens. As a non-replicative subunit vaccine, ESX1-Vax is also readily boostable. Furthermore, administration of ESX1-Vax/CAF01 in a post-exposure setting is highly efficacious against bacterial re-growth. In this proposal, we aim to optimize the immunogencity and efficacy of this ESX1-Vax through molecular modifications to maximize the T cell response against ESAT-6 epitopes. We will compare the modified constructs for efficacy and immunogenicity after BCG co-vaccination and in standard and newly-established post-exposure vaccination models to select the optimal ESX1-Vax construct. The selected construct will then be developed for production as a final GMP-produced ESX1/CAF01 product, verified for vaccine activity and assessed for toxicology. The overall outcome of this product development is, thus, a single boostable vaccine that is uniquely designed to work both together with BCG prophylactically in non-infected neonates, as well as when delivered post Mtb exposure in latently infected individuals, and that is ready for progress to clinical trials.